Several distinct cytotoxins can be isolated from any given cobra venom sample, and more than 50 variants have been sequenced (Dufton and Hider, 1991). These cobra cytotoxins are polybasic, yet hydrophobic proteins, typically consisting of 60 amino acids, with a molecular weight of 6000–7000 Daltons. Crystallographic and nuclear magnetic resonance (NMR) analysis indicates that these proteins contain no alpha helical structure, but rather consist of three loops of anti-parallel beta strands emanating from a core that is constrained by four disulfide bonds, as shown in FIG. 1. Structural features thought to be responsible for their activity involve rather flat beta sheet regions of hydrophobic residues flanked by cationic amino acids (Kini & Evans, 1989). These toxins induce rapid depolarization of skeletal and cardiac muscle. Some of the toxins are selectively cytotoxic for a variety of human cancer cell lines. Previous comparisons of native toxins indicated that Naja naja atra III cytotoxin (hereinafter abbreviated as CTX) was highly toxic toward human leukemic T-lymphocytes, whereas the toxin from Naja naja oxiana displayed almost no toxicity, even though the two proteins differed at only 4 of their 60 residues. Moreover, two of these differences occurred in the first loop (Stevens-Truss & Hinman, 1997).
Chemical modifications of individual amino acids have contributed toward an elucidation of structural features that may underlie cytotoxicity. After separation of toxins modified by acetylation of individual lysine residues, Lin, Chang & Chang (1993) and Gatineau et al. (1990) reported that the most critical lysine was at position 12 in the first loop of cytotoxin. Earlier, Gatineau et al. (1987) using nitration of tyrosine, indicated that the tyrosine at position 11 in Loop 1 was the most important of the three tyrosines in the molecule. From other studies, it has been suggested that cardiotoxicity, as opposed strictly to cytotoxicity, depends upon amino acid residues in loops 2 and 3.
Previous work by the inventors herein involving synthetic analogs of each of the three loops of cytotoxin III from Naja naja atra has shown than a Loop 2 construct, in which the Cys at position 21 forms an unusual disulfide bond with the Cys at position 38 (FIG. 1), had no activity against either heart cells or against human tumor T-lymphocytes (the CEM line). By contrast, the Loop 1 analog, in which the Cys at position 3 is constrained to form a disulfide bond with the Cys at position 14 (FIG. 1), was highly active in its ability to inhibit thymidine incorporation in CEM tumor cells.
Two prior patents have been found that involve components of cobra venom. The first, Vidal, U.S. Pat. No. 5,232,911 issued in 1993, utilized a whole cardiotoxin reported to have been purified from N. n. atra, but whose indicated sequence has an extra alanine within the first 15 residues and which more closely resembles toxins from Naja haje or from Naja nivea (Dufton and Hider, 1991). This 61-amino acid protein was for use in cancer chemotherapy strictly as an enhancer of the phospholipase activity of a heterodimer isolated from another snake, Crotalus durissus terrificus. The only application of the N. n. atra peptide was when it would be administered in combination with the much larger heterodimer. The Lipps et al. U.S. Pat. No. 5,565,431 issued in 1996, involved a toxin isolated from the Thailand cobra, Naja naja Kaouthia, which the inventors termed “Kaotree.” Having a reported molecular weight based on gel electrophoresis of approximately 6000 Daltons, the sequence of its first 15 amino acids [using single-letter abbreviations: MECYRMSNIVTCQPW] Seq. ID No. 19, is very much different from the sequence of any of the cardiotoxins from N. n. siamensis (Kaouthia) [or for any of the 50+ cardiotoxins reported by Dufton and Hider (1991)].